Ultrasound-activated piezo-hot carriers trigger tandem catalysis coordinating cuproptosis-like bacterial death against implant infections

Implant-associated infections due to the formation of bacterial biofilms pose a serious threat in medical healthcare, which needs effective therapeutic methods. Here, we propose a multifunctional nanoreactor by spatiotemporal ultrasound-driven tandem catalysis to amplify the efficacy of sonodynamic and chemodynamic therapy. By combining piezoelectric barium titanate with polydopamine and copper, the ultrasound-activated piezo-hot carriers transfer easily to copper by polydopamine. It boosts reactive oxygen species production by piezoelectrics, and facilitates the interconversion between Cu2+ and Cu+ to promote hydroxyl radical generation via Cu+ -catalyzed chemodynamic reactions. Finally, the elevated reactive oxygen species cause bacterial membrane structure loosening and DNA damage. Transcriptomics and metabolomics analysis reveal that intracellular copper overload restricts the tricarboxylic acid cycle, promoting bacterial cuproptosis-like death. Therefore, the polyetherketoneketone scaffold engineered with the designed nanoreactor shows excellent antibacterial performance with ultrasound stimulation and promotes angiogenesis and osteogenesis on-demand in vivo.

Coordinating Cuproptosis-Like Bacterial Death Against Implant Infections".We do appreciate the valuable comments and suggestions from the reviewers, which improve the quality of our manuscript.We have carefully revised the manuscript according to the reviewers' suggestions.
The annotations stated in this response letter and revisions made in the Revised Manuscript are listed as follows: Reviewer #1

Comments:
The manuscript introduced a strategy utilizing US-activated tandem catalysis to amplify the therapeutic effect of SDT and CDT for the on-demand treatment of IAI via CpBT nanoreactors.
The authors did a decent job in material design and biological experiments.However, the following issues must be clarified before accept this manuscript.
Answer: Thanks very much for your affirmation and comments.We have revised our manuscript and responded to your questions carefully.Specific explanations are shown below.
1.The temperature dependence of relative permittivity of the dielectric constant of CpBT is missing in Fig. 2e.
Answer: Thank you for your comment.The temperature dependence of the dielectric constant of CpBT was shown in Figure R1.The dielectric properties showed diffused phase transition at the temperature range of 20 to 45 o C due to the presence of polymer component pDA.In this work, the core mBT acted as an electron provider under US stimulation, and the shell pDA acted as an "electron aspirator" to transport electrons to Cu 2+ .Therefore, the dielectric and piezoelectricity properties of mBT were more important.In the manuscript, we paid more attention to the dielectric properties of mBT, to clarify that in the body temperature mBT showed better piezoelectric performance than pure BT, which was in accordance with Landau free energy modeling.

Figure R1
The temperature dependence of dielectric constant of CpBT measured at 1 kHz.
2. In Fig. 2f, the authors compare the temperature dependence of the dielectric constants of BT and mBT.The free-energy profiles of BT and mBT at different temperatures should be added and discussed to highlight the advantages of mBT.
Answer: Thank you for your comment.We have conducted the Landau free energy modeling of pure BT at different temperatures (Figure R2).The pure BaTiO3 in the T phase showed large polarization anisotropy energy (> 1 J/cm 3 ) in the temperature range of 25-43 o C.However, in the same temperature range, mBT showed significantly decreased polarization anisotropy with a small energy gap among O and T phases (Figure R3) (< 0.3 J/cm 3 ), favoring the higher piezoelectricity.
Figure R2 The free-energy profiles for pure BT at different temperatures.

Figure R3
The free-energy profiles for mBT at different temperatures.
We have added the Landau free energy profiles of pure BT at 37 °C to Figure 2f, and more discussion has been made in the Revise Manuscript on page 7, line 22: "Additionally, the Landau free energy modeling revealed that at temperature of 37-43 °C, the polarization anisotropy energy and energy barrier (< 0.3 J/cm 3 ) were relatively lower than that of pure BT (> 1 J/cm 3 ) (Fig. 2f, Supplementary Fig. 3 and Fig. 4), which led to a small energy barrier for polarization rotation among T <100> and O <110> states 26 , hence inducing the enhanced piezoelectric performance over the body temperature." 3. In Fig. 2h, the peaks of Cu + and Cu 2+ in Cu 2p orbitals should be analyzed and the specific ratio of different valence states of copper before and after the reaction should be added as mentioned in the article.In addition, the colors of fresh and 934.9 eV are too similar and should be replaced.
Answer: Thank you for your comment.The peaks of Cu + and Cu 2+ in Cu 2p orbitals before reaction had been shown in Supporting Information before.Now, we have added it to Figure 2i, and the color of the peak at 934.9 eV had been replaced.It showed that the higher peak at ∼935 eV in Cu 2p3/2 spectra was assigned to Cu 2+ , accompanied by the characteristic Cu 2+ shakeup satellite peaks (938-945 eV).The lower peak at ∼932 eV suggested the presence of Cu + or Cu 0 species.Furthermore, the Cu LMM Auger spectra at ~570 eV confirmed the presence of Cu + after US.Notably, the integral area ratio of Cu + to Cu 2+ after US was significantly enhanced at 935 eV (from 0.28:1 for fresh CpBT to 0.67:1 for used CpBT) and at 570 eV (from 0.33:1 for fresh CpBT to 0.5:1 for used CpBT) (Fig. 2j), indicating that part of surface Cu 2+ species were reduced to Cu + species during US stimulation.The real space scattering states of the 16-layer stacked DHI and DA system at different energy levels.
All the figures share the same color bar.
In manuscript, page 11, line 7: "Interestingly, the transmission spectra (Fig. 3c and 3d) revealed that the transmission coefficient near HOMO and LUMO was zero, and that transmission cannot occur at these energy levels, consistent with the LOL-π isosurfaces observations in the layered aggregation structure that there was no overlap of interlayer charge densities (Supplementary Fig. 7)." in the Results and discussions section was changed to "Notably, the transmission spectra (Fig. 3c) demonstrated a zero transmission coefficient near the HOMO and LUMO, with no observable opened transport channel in the real-space scattering states below the LUMO energy level (< 0.1 eV, 2 in Fig. 3d).The absence of transmission around the HOMO and LUMO energy levels concurred with the observations of a non-overlapping interlayer charge density in LOL-π isosurfaces within the layered aggregation structure."

5.
The article mentions that intracellular copper overload restricts the tricarboxylic acid cycle and promotes bacterial death by copper toxicity, so is it also cytotoxic in that state.
Meanwhile, it is mentioned that the CpBT nanoreactor can cause DNA and protein leakage, but there is a lack of relevant proof.The bacterial protein leaking from S. aureus after incubation on different scaffolds was determined by the Micro BCA protein assay reagent kit, as shown in Figure R5a.PH-CpBT with US stimulation had much higher total proteins than other groups, confirming that cell membranes were damaged seriously.Besides, the total intact bacterial DNA was also obtained by Bacterial DNA Kit for different groups, and the quantitative data was shown in Figure R5b.
Of note, only 20% of intact DNA was detected in PH-CpBT+H2O2+US group, suggesting that bacterial DNA was gravely devastated by the US-initiated ROS storm, which was consistent with transcriptomics (Fig. 4g-k).We have added more discussion to the Manuscript on page 15, line 4: "The leaking of bacterial proteins was detected using the protein leakage assay, and PH-CpBT exhibited the highest protein leakage, indicating significant membrane permeability alteration and cytoplasmic leakage after US-activated tandem catalysis (Supplementary Fig. 22a).Besides, the total bacterial DNA was quantified by Bacterial DNA Kit for different groups (Supplementary Fig. 22b).Notably, only a few intact DNA was detected in PH-CpBT+H2O2+US, indicating severe damage to bacterial DNA due to the initiated ROS storm." About Cu-related cytotoxicity, more discussion has been added to page 22, line 3: "To verify cell adhesion and proliferation, various scaffolds were cultured with MC3T3-E1 cells in vitro.Cell viability by CCK-8 assay showed almost no cytotoxicity in all scaffolds (Supplementary Fig. 29).Besides, all scaffolds except for P and Pp showed similar proliferation rates (Supplementary Fig. 30).With 3 days of culture, cells efficiently infiltrated the entire volume of PH-CpBT, and their actin cytoskeleton exhibited an elongated morphology throughout the scaffold, indicating good biocompatibility and cell adhesion (Supplementary Fig. 31)." 6.In Fig. 4d, the graph only gives US and US+H2O2+CpBT, and does not prove the effect of H2O2 alone on bacteria, the conclusion given in this experiment is not accurate enough.
Answer: Thank you for your comment.As the reviewer suggested, to explore the influence of H2O2, we included H2O2+US and CpBT+US groups, as shown in Figure R6.It can be observed that when bacteria with the addition of H2O2 was subjected to US stimulation, both S. aureus and E. coli maintained intact morphology.In contrast, CpBT+US group exhibited only partially blurry bacterial membrane structures.In the presence of bacteria, the elevated levels of H2O2 in the vicinity were a result of the host's defense mechanism against the bacteria [Clin.Micro.Rev.
1997, 10(1): 1-18].In order to simulate the elevated H2O2 environment in the infected microenvironment, we utilized the same concentration of H2O2 as in previous studies (Angew. Chem.Int.Ed. 2023, 62, e2022104).Consistent with their findings, our study also concluded that the involvement of H2O2 alone will not result in significant structural damage to bacterial cells.We have added more discussion to the Manuscript on page 14, line 8: "To observe microscopic changes in dead bacteria, Bio-TEM images of bacteria were examined (Supplementary Fig. 17a).Bacteria incubated with PH-CpBT+H2O2+US showed incomplete walls and cytoplasmic leakage in both E. coli (Fig. 4d) and S. aureus (Fig. 4e), and bacteria with H2O2+US maintained intact morphology." 7. The genes in Fig. 4g and 4h that are significantly different and associated with biofilm elimination should be labeled.
Answer: Thank you for your comment.To comprehensively understand the changes in biofilmrelated genes across the three groups, we marked these genes on the heatmap (Fig. 4k).This visualization enabled a clearer examination of the differences in gene expression levels among the three groups.It was evident that PH-CpBT significantly interfered with biofilm formation.We have added more discussion to the Manuscript on page 14, line 7: "To observe microscopic changes in dead bacteria, Bio-TEM images of bacteria were examined (Supplementary Fig. 17a).Bacteria incubated with PH-CpBT+H2O2+US showed incomplete walls and cytoplasmic leakage in both E. coli (Fig. 4d) and S. aureus (Fig. 4e), and bacteria with H2O2+US maintained intact morphology.Besides, element mapping of bacteria showed intracellular copper content was distinct in PH-CpBT+H2O2+US compared with US+H2O2 and PH-CpBT+H2O2 (Supplementary Fig. 17b), indicating an increased intracellular copper inward flow due to the membrane permeability alteration induced by evaluated ROS (Fig. 4f and Supplementary Fig. 18).Previous reports showed that Cu ions have dose-dependent antibacterial activity 22 .To find out the antibacterial effect of Cu ions in this work, Cu ions released from PH-CpBT scaffolds with or without US stimulation were analyzed by an ICP spectrometer (Supplementary Fig. 19).The total amount of released Cu ions was 3-7 μg/L, much lower than MIC of Cu 2+ ions (630 ug/L for S. aureus and 63-630 ug/L for E. coli) 32 .
Furthermore, S. aureus or E. coli.were co-incubated with the leaching solution of PH-CpBT after US stimulation (30 min) for 24 h (Supplementary Fig. 20a), finding that the antibacterial rate was lower than 50%, indicating that the antibacterial performance by only released Cu ions was poor.Besides, bacteria were incubated with PH-CpBT+US and tetrathiomolybdate (TTM, a copper chelator).In this situation, Cu ions cannot get into the bacteria, resulting in an antibacterial rate of 70% due to ROS attack (Supplementary Fig. 20b).Furthermore, mBT and Cu ions coating on Pp without pDA as interlayer exhibited a lower antibacterial rate than PH-CpBT (Supplementary Fig. 20c), suggesting that the electron transport within pDA was a key factor for the reduction of Cu 2+ .These results proved that the antibacterial mechanism was more  We have added more discussion to the Manuscript on page 15, line 1: "In addition, we compared the antibacterial activity of CpBT with other piezoelectric materials (including BT, ZnO, MoS2 and TiO2), finding that CpBT showed better antibacterial activity (Supplementary Fig. 21)." 10.In the in vivo experiments, the authors did not record the weight of each group of mice statistically, so there is no guarantee that each group of mice is basically the same.
Answer: Thank you for your comment.Initially, rats weighing approximately 250 g were procured for our study.They were randomly caged and underwent a about two-week acclimatization period before the commencement of the experiments.Subsequently, during the experimental grouping, rats from the same cage were evenly distributed across various test groups to ensure baseline uniformity.Additionally, we measured weight before experiment, confirming no significant differences in the initial weights among groups (Figure R9).
Figure R9 The initial body weight of rats for different groups.
We have added more discussion to the Manuscript on page 19, line 19: "The baseline weights of all groups were equivalent before surgery (Supplementary Fig. 27)." 11.It is suggested that the relevant content of the H&E images should be labelled, such as lymphocytes, and the quantitative analysis should be added for better observation and understanding by readers.
Answer: Thank you for your comments and suggestions.The nuclei of neutrophils appeared dark purple, and the cytoplasm appeared pale pink.For better observation, the neutrophils and lymphocytes were marked with red and green arrows, respectively (Fig. 5d).And the related quantitative analysis was shown in Fig. 5e.12. Statistical analysis should be added to the CT range area in Fig. 6a to visualize the speed of new bone growing.
Answer: Thank you for your comment.On one hand, we demonstrated the growth rate and overall quantity of new bone in PH-CpBT group at different stages through videos (Supplementary Information, Video 1), where the red portions represented newly formed bone.
On the other hand, in Fig. 6a, we incorporated the amount of new bone at each stage, providing a clearer depiction of the new bone growth rates at 4 and 8 weeks.13.DAPI is a DNA dye that clearly shows the nucleus and thus the distribution of cells, and should be the largest of all stains, while the range of DAPI in the stained picture in Fig. 7d is too small Answer: Thank you for your reminding.In the previous version, DAPI color was overlooked to emphasize RUNX2, BMP-2, and CD31.Indeed, in the overall staining, DAPI should exhibit the strongest expression, even in Pp group.We have updated the images in Fig. 7d to rectify this, and the alterations do not affect the differences between the groups in terms of osteogenesis and angiogenesis.1.The author mentioned that the Cu 2+ and single SDT is not active.However, numerous studies have demonstrated the potential of SDT in both tumor and bacterial eradication.
Additionally, Cu 2+ ions are widely recognized for their antibacterial properties in tissue engineering.Therefore, the authors should provide a more compelling explanation in their manuscript, addressing my primary concern: whether the notable antibacterial effect arises from an enhanced release of Cu 2+ ions induced by ultrasound.
Answer: We are grateful to the reviewer for their meticulous examination of our manuscript and their insightful comments.We are sorry that we haven't expressed the word "inactive" correctly.In this work, the word "inactive" in Fig. 1  ).Besides, we admitted that antibacterial performance of Cu 2+ ions is quite good, but it depends on the concentration, which was low in our work (please see below).To avoid misunderstanding, the word "inactive" in Fig. 1 was placed by "inefficient".
Besides, we fully agreed that it was critical to demonstrate the antibacterial effect of the released Cu ions from PH-CpBT scaffold by ultrasound, and we thank the reviewer for pointing it out.A series of experiments were conducted to clarify this issue: Firstly, Cu 2+ ions released from PH-CpBT scaffolds at 1-, 4-, and 7-day intervals and with or without US stimulation (10 min/day, 1 MHz, 1.0 W/cm 2 , 50% duty cycle, the same as in vivo treatment parameters) were analyzed by an inductively coupled plasma (ICP) spectrometer.
Specifically, PH-CpBT scaffolds were immersed in a 0.9 % NaCl solution at 37 ± 1 °C with the surface-area to -volume ratio was 3 cm 2 /mL according to international standard ISO 10993-12.
Triplicate samples were used to obtain an average value with standard deviation.As shown in   Secondly, to explore the antibacterial performance of released Cu 2+ in this work, we did a series of antibacterial experiments (Figure R2).Firstly, S. aureus or E. coli.were co-incubated with the leaching solution of PH-CpBT scaffold after US stimulation (30 min) for 1 day (Figure R2a), finding that the antibacterial rate was lower than 50% (Figure R2e-g R2b) under US stimulation.In this situation, Cu ions cannot get into the bacteria, so the antibacterial effect only came from ROS production by tandem catalysis between SDT and CDT, representing an antibacterial rate of ~70%, which indicated that Cu-related cuproptosis-like bacterial death was also important antibacterial mechanism in this work.
At last, in CpBT system, pDA served a crucial function as an electron transporter in the conversion of Cu ions.Therefore, Pp scaffold was coated with mBT and Cu ions, whose content was equal to CpBT on PH-CpBT scaffold (Figure R2c, defined at PH-mBT/Cu group).In this situation, the antibacterial effect originated from ROS generated by SDT of mBT, and Cu 2+ release with US stimulation.The antibacterial rate of PH-mBT/Cu was ~75%, indicating that pDA, which acted as the "electron aspirator" to extract US-activated piezo-hot carriers and initiated the oxidizing reaction of Cu 2+ to Cu + , played an important role in this work.
As a result, apart from Cu 2+ release, US-activated piezo-hot carriers trigger tandem catalysis played an important part in antibacterial performance, and increased copper accumulation occurred only under the influence of ROS, contributing to cuproptosis-like bacterial death caused by copper."To observe microscopic changes in dead bacteria, Bio-TEM images of bacteria were examined (Supplementary Fig. 17a).Bacteria incubated with PH-CpBT+H2O2+US showed incomplete walls and cytoplasmic leakage in both E. coli (Fig. 4d) and S. aureus (Fig. 4e), and bacteria with H2O2+US maintained intact morphology.Besides, element mapping of bacteria showed intracellular copper content was distinct in PH-CpBT+H2O2+US compared with US+H2O2 and PH-CpBT+H2O2 (Supplementary Fig. 17b), indicating an increased intracellular copper inward Besides, the characteristic absorption peak increased slowly with US time increased, suggesting that the production of Cu + was time-dependent (Supplementary Fig. 6a).Without piezoelectrons produced by mBT, the reduction of Cu 2+ to Cu + under US stimulation failed (Supplementary Fig. 6b and c)." 3. In the process of preparing the coated scaffold, the authors solely employ the method of solution soaking.This prompts the question of whether the antibacterial effect primarily results from ion release in a water-based environment.The stability of the coating under such conditions warrants investigation.
Answer: We thank the reviewer for the careful reading of our manuscript.As we answered in ).The decoration of PDA films on these materials not only improves their solubility and stability but also renders them multifunctional and smart platforms.
Furthermore, supposed that the pDA coating was not that stable, CpBT nanoreactor would fall off PH-CpBT scaffold, along with the released Cu ion from CpBT, as shown in Figure R4 (process A).We collected the leaching solution of PH-CpBT after US (10 mins/day) for 7 days,  4. Furthermore, the authors have only presented the antibacterial properties of the powder form of their material.It is essential to assess the catalytic ability of the scaffold itself, as this will provide a more comprehensive understanding of its antibacterial potential.
Answer: We thank the reviewer for careful reading of our manuscript.All the antibacterial experiments in this work were carried out with scaffolds.Taking the antibacterial plate experiment as an example, following the scaffold preparation method outlined earlier, we obtained three scaffolds for each group.Subsequently, the scaffolds were placed into bacterial suspensions at a scaffold surface area/volume ratio of 1.5.After US stimulation for 10 mins, an appropriate bacterial suspension was taken for plate observation.ions in this work, we synthesized pDA nanoparticles, and the same amount of Cu 2+ was chelated to the surface of pDA (pDA@Cu).Then, we used the specific reagent neocuproine to detect whether the valence state of Cu 2+ on pDA@Cu will change under US.The result was shown in Figure R5a, the characteristic absorption peak of the [Cu(neocuproine)2] + did not increase as time went by, suggesting that the conversion of Cu 2+ to Cu + failed in absence of piezoelectric mBT.Besides, we mixed free Cu 2+ (from CuSO4) with neocuproine, and the color of solution also did not change, indicating that low-intensity ultrasound (1.0 W/cm 2 , 50% duty, 1 MHz) cannot change the valence state of Cu 2+ (Figure R5B).It can draw a conclusion that piezoelectricity of mBT that showed SDT performance played a crucial role in this process.R3).The decreased metabolites negatively regulated the bacterial system, leading to an upregulation of more TCA cycle genes.This also demonstrated that copper does not directly affect genes but first influences lipoylation proteins, thereby impacting the entire bacterial metabolic pathway.We have added more discussion to the Manuscript on page 16, line 27:

Additionally
"To present the changes in metabolites and the relevant genes in the TCA cycle more clearly, we conducted targeted detection of TCA cycle metabolites.We observed the lowest levels of metabolites in PH-CpBT+H2O2+US compared with Control and PH-CpBT+H2O2 (Supplementary Fig. S25).Fig. 4l vividly illustrated the changes in TCA cycle metabolites and the genes influenced by them.In short, under the influence of ROS, bacteria with PH-CpBT+H2O2+US experienced an increase in intracellular Cu, which bound with four lipoylation enzymes (DLAT, GCSH, DBT, and DLST) 19,34 , leading to their deactivation and consequent reduction in metabolites throughout the pathway.Additionally, due to the formation of more lipoylation proteins, lipoic acid was reduced 35 , resulting in the upregulation of the genes that regulated lipoic acid synthesis 36 .Ultimately, the decreased metabolites, through negative feedback regulation, increased the expression of genes regulating the TCA cycle.Therefore, through metabolomics and transcriptomics, it was evident that copper-induced cuproptosis-like death, causing an overall reduction in metabolism, and in synergy with ROS, effectively killed bacteria." With these modifications, we hope now the manuscript is acceptable for publication in Nature Communications.

Detailed
Response to the Reviewers' Comments (No. NCOMMS-23-37813A) Dear Reviewers, Thank you for the constructive and insightful comments on our manuscript (No.NCOMMS-23-37813A) entitled "Ultrasound-Activated Piezo-Hot Carriers Trigger Tandem Catalysis

Fig. 2 i
Fig. 2 i XPS of Cu 2p3/2 for CpBT before and after US stimulation (the insets show Cu LMM spectra).iPeak ratio of Cu + to Cu 2+ at 570 eV and 932 eV in XPS spectra of CpBT before and after US stimulation.

Fig 3 .a
Fig 3. a Energy levels and b the corresponding density of states (DOS) of 16-layer stacked DHI and DA.c The transmission spectra of the 16-layer stacked DHI and DA-based transport architecture device.d

Answer:
Thank you for your comment.In the human body, copper served a diverse range of functions.Under normal physiological conditions, intracellular copper concentrations were kept at extraordinarily low levels by active homeostatic mechanisms that worked across concentration gradients to prevent the accumulation of free intracellular copper that was detrimental to cells [Nat.Chem.Bio., 2008, 4(3): 176-185; Nat.Rev. Cancer, 2022, 22(2): 102-113].To figure out whether Cu ions had toxicity in this work, Cu ions released from PH-CpBT scaffolds at 1-, 4-, and 7-day intervals and with or without US stimulation were analyzed by an inductively coupled plasma (ICP) spectrometer according to the international standard ISO 10993-12, as shown in Figure R4a.It showed that PH-CpBT scaffold released Cu ions at a concentration of 3-7 μg/L with or without US stimulation.The cytotoxicity of Cu ions was closely related to the concentration and cell lines, and

Figure R4 a
Figure R4 a Cu ions released from the PH-CpBT scaffold in 0.9 % NaCl solution with different immersion time and US stimulation at 37 °C.b Cytotoxicity of different scaffolds to MC3T3-E1 cells in this work.

Figure R5
Figure R5 Quantitative analysis of a protein leakage and b the intact bacterial DNA of S. aurues on different scaffolds after different treatments.

Figure R6
Figure R6 TEM images of bacteria after different treatments.

Fig. 4 k
Fig. 4 k Left, heat map showing the differential expression of genes of interest.Right, the death pathways by ROS-induced oxidative stress and Cu-induced cuproptosis-like death.

Fig. 5
Fig. 5 The treatment of implant infection with PH-CpBT in vivo.a Schematic illustration of infected modified PEKK scaffolds and animal experimental treatment in therapeutic.b Photographs of bacterial colonies and turbid liquid.c Quantitative analysis of bacterial turbid liquid by OD 600.d H&E images and e semi-quantification of neutrophil in the infected bone tissues surrounding the implants.The red arrows represented neutrophils, and the green arrows represented lymphocytes.f Giemsa staining images and g semi-quantification of bacteria in the infected bone tissues surrounding the implants.The

Figure
Figure R7 a-d Schematic diagram for different antibacterial experiments.e Typical images of S. aureus and E. coli colonies treated by various groups.The corresponding antibacterial rate against f S. aureus and g E. coli after different treatments.The experiment was repeated three times independently, with a representative example shown.Significant differences between groups were indicated as ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.
intricate than the release of Cu ions."Besides, we added additional animal experiments to clarify the role of Cu ions in vivo to the Revised Manuscript on page 19, line 24: "Visual examination revealed the presence of secretions and pus at the implant site of Pp and PH, with partial mitigation observed in PH-pBT, PH-CpBT (US-), and PH-mBT/Cu (Supplementary Fig.28).By comparison, the implant site of PH-CpBT+US and vancomycin (Van.)exhibited smooth tissue healing without secretion and pus formation, suggesting the clearance of bacterial infection.The order of bacterial colonies on agar plates and the turbidity of the Luria-Bertani medium after cultivation were as follows: Pp ≈ PH > PH-pBT ≈ PH-CpBT (US-) > PH-mBT/Cu > PH-CpBT ≈ Van, suggesting that PH-CpBT exhibited favorable in vivo antibacterial characteristics rivaling Van (Fig.5b and 5c).For PH-mBT/Cu, the lack of pDA as "electron aspirator" to initiate the reduction of Cu + , the antibacterial performance by SDT and released Cu 2+ only was inefficient.For PH-pBT, no induction of copper-induced cuproptosislike bacterial death occurred due to the absence of Cu ions, also exhibited moderate antibacterial effect."9.The antimicrobial performance or biofilm elimination ability of this nanoreactor CpBT should be compared with similar materials to demonstrate the advantages of this material.Answer: Thank you for your comment.To compare the antibacterial performance of CpBT with similar materials, including commercial pure BaTiO3 (Macklin Inc.), ZnO (Macklin Inc.), MoS2, and TiO2 (Sinopharm Chemical Reagent Co., Ltd.) were subjected to the antibacterial experiment with US stimulation (FigureR8).The first three were classic piezoelectric material (Adv.Mater.2019, 31, 1802084), and the last was sonosensitizer that was often investigated in SDT (J.Am.Chem.Soc.2020, 142, 6527−6537).The particle size of above materials was about 100 nm.It can be seen that although these groups showed certain antibacterial performance, with the antibacterial rate lower than 70%.Obviously, CpBT showed better antibacterial properties due to ultrasound-activated piezo-hot carriers triggering tandem catalysis coordinating cuproptosis-like bacterial death.

Figure R8 a
Figure R8 a Plate cultures of S. aureus and E. coli treated with different materials and US stimulation.The related antibacterial rate against b S. aureus and c E. coli.

Fig. 5 d
Fig. 5 d H&E images and e semi-quantification of neutrophil in the infected bone tissues surrounding the implants.The red arrows represented neutrophils, and the green arrows represented lymphocytes.

Figure R10
Figure R10 Typical screenshots of bone generation video.

Fig. 6
Fig. 6 The number and speed of new bone growing into the scaffold.a Mico-CT of femoral condyle, from up to down, reconstruction of defect and new bone ingrowth in the scaffolds, top view of new bone, side view of new bone.Quantitative statistics of bone regeneration related index in 3D reconstruction by micro-CT including.

Figure R1a ,
FigureR1a, the total amount of released Cu 2+ ions were 3.4, 4.0, and 4.3 μg/L after 1, 4, and 7 days of immersion, respectively.The ultrasound stimulation will accelerate the release of Cu 2+ ions, reaching 4.6, 6.3, and 7.3 μg/L at 1, 4, and 7 days.It was reported that the antimicrobial minimum inhibitory concentration (MIC) of Cu 2+ ions was 630 ug/L for S. aureus and 63-630 ug/L for E. coli.(Chem.Res.Toxicol.2015, 28, 1815-1822), which was more than 10 times higher than the release concentration of Cu 2+ in this work.Besides, for antibacterial Cucontained metal or alloys, we surveyed the existing scientific literature and statistically integrated the findings, and the relationship between the antibacterial rate and Cu 2+ ion release concentration was shown in FigureR1b.It showed that the low Cu 2+ release concentration (<10 ug/L) resulted in bad antibacterial rate (<70%), and good antibacterial performance (>90%) required higher Cu 2+ concentration (>100 ug/L).However, in this work, the low Cu 2+ release concentration from PH-CpBT scaffolds (<8 ug/L with US stimulation) resulted in good antibacterial rate (99.95%).Therefore, apart from Cu 2+ release, US-activated piezo-hot carriers triggering tandem catalysis played an important part in antibacterial performance.Besides, the enrichment of Cu ions in bacteria was revealed by examining the ultrastructure of the bacteria using Bio-TEM (FigureR1c).Compared with US+H2O2 and PH-CpBT+H2O2 group,

Figure R1 a
Figure R1 a Cu 2+ released from PH-CpBT scaffold in the 0.9 % NaCl solution at different immersion time with or without US stimulation.b Summary of antibacterial rates and Cu ions released concentrations of the reported antibacterial alloys (Mat.Sci.& Eng.C 2020, 115, 1; Biomed.Mater.

Figure
Figure R2 a-d Schematic diagram for different antibacterial experiments.e Typical images of S. aureus and E. coli colonies treated by various groups.The corresponding antibacterial rate against f S. aureus and g E. coli after different treatments.The experiment was repeated three independently, with a representative example shown.Significant differences between groups were indicated as ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.

Figure R3 a
Figure R3 a The reaction mechanism of Cu + detection by neocuproine.b UV-Vis absorbance spectra of neocuproine solution treated with CpBT and US stimulation.
and detected the concentration of Cu ions by ICP was ~8 ug/L.n process B, we used the centrifugal machine to exclude CpBT nanoreactor in the leaching solution, and the concentration of Cu ions detected by ICP was ~7 ug/L.The concentration of Cu ions in two processes was similar, indicating that the content of CpBT nanoreactor in leaching solution was low.The results suggested that the pDA coating was stable with US stimulation.

Figure R4
Figure R4 Cu ions released from PH-CpBT scaffold with different treatments.

Figure
Figure R5 a UV-Vis absorbance spectra of pDA@Cu and b Cu 2+ ions with US stimulation.

Figure R2
Figure R2 Quantitative analysis of a protein leakage and b the intact bacterial DNA of S. aurues on different scaffolds after different treatments.

Figure R3 A
Figure R3 A correlation analysis between differential metabolites and differentially expressed genes.Red rectangles indicated increased metabolites, and green rectangles indicate decreased metabolites; Red circles indicated up-regulated genes, and green ovals indicated down-regulated genes.DBT: Dihydrolipoamide Branched Chain Transacylase E2, GCSH: Glycine Cleavage System Protein H, DLST: Dihydrolipoamide S-Succinyltransferase, DLAT: Dihydrolipoamide S-Acetyltransferase.
), indicating that the antibacterial performance by released Cu ions only was not that good.
Q1, the released concentration of Cu ions from PH-CpBT scaffold is quite low.PEKK possesses good surface chemical modification potential, which is easier to modify.In this work, PEKK scaffolds were modified with polydopamine (defined as Pp scaffolds), then Pp scaffolds were further modified with CpBT.Polydopamine (pDA) coating of surfaces was a versatile strategy to fabricate functional films on various substrates.As a mussel-inspired material, polydopamine, which possessed many properties, such as a simple preparation process, good biocompatibility, strong adhesive property, and easy functionalization, can easily functionalize broader range of surfaces, including most noble metals and metal oxides as well as materials with low surface energy (titanium alloy, carbon nanotubes, graphene, PS, polyether-ether-ketone etc.) (Nat. , some studies also reported the potential for ultrasound to facilitate the conversion of Cu 2+ ions into Cu + ions.Consequently, it is imperative to reconsider and conduct further experiments to explore the relevance of SDT and CDT in this work.How important role of SDT play in this process?Answer: Thank you for your comment.To our knowledge, the conversion of Cu 2+ to Cu + in vivo was mostly realized by glutathione (enrichment in tumor tissue) (J.Am.Chem.Soc.2019,NanoLett.2019,19,1749-1757).There were few reports on in situ conversion of Cu2+to Cu + triggered by low-intensity ultrasound that applied in this work.Pedro et al. reported that ultrasound-assisted Cu + -catalyzed alkyne-azide click reaction by metallic copper [Nat.Protoc.2010,5(3),607], and the ultrasound used in this work was high-power US probe system (21 kHz, 25 W) working at higher temperature of 70 °C or 100 °C.Another work by Chen et.al.reported that ultrasound-driven bioorthogonal catalytic therapy through ultrasmall poly(acrylic acid)-modified copper nanocomplexes (Cu@PAA NCs) (Adv.Mater.2023,35, 2209179).They proposed that the electrons can be ejected from the surface of Cu@PAA NCs through the photoelectric effect by US-mediated sonoluminescence, which converted Cu 2+ to Cu + .The conversion mechanism that related to photo-electrons was different from our work that the conversion of Cu 2+ to Cu + in our work was triggered by US-induced piezo-hot carriers.Besides, to figure out whether ultrasound can facilitate the conversion of Cu 2+ into Cu + Sincerely, Prof. Zongke Zhou, Director, Department of Orthopaedics, Orthopaedic Research Institute West China Hospital, Sichuan University, China Email: zongke@126.comzhouzongke@scu.edu.cnProf. Xianzeng Zhang Dean, College of Photonic and Electronic Engineering Fujian Normal University, China Email: xzzhang@fjnu.edu.cn